Detectable molecules, method of preparation and use

ABSTRACT

A detectable molecule of the formula 
     
         A.sup.3 --(--X--R.sup.1 --E--Det.sup.b).sub.m 
    
     where A 3  is A 2  or a polymer, where A 3  has at least one modifiable reactive group selected from the group consisting of amino, hydroxy, cis .OH, halides, aryl, imidazoyl, carbonyl, carboxy, thiol or a residue comprising an activated carbon; --X-- is selected from the group consisting of ##STR1## a C 1  -C 10  branched or unbranched alkyl or aralkyl, which may be substituted by --OH; --Y-- is a direct bond to --E--, or --Y-- is --E--R 2  -- where R 2  is a C 1  -C 10  branched or unbranched alkyl; Z a  is chlorine, bromine or iodine; E is O, NH or an acylic divalent sulfur atom; Det b  is a chemical moiety capable of being detected, preferably comprising biotin or a metal chelator of the formula: ##STR2## or the 4-hydroxy or acyloxy derivative thereof, where R 3  is C 1  -C 4  alkyl or CH 2  COOM, M is the same or different and selected from the group consisting of hydrogen, a metal or non-metal cation or is C 1  -C 10  alkyl, aryl or aralkyl; and m is an integer from 1 to the total number of modified reactive groups on A 3 . The detectable molecules are useful in in vitro or in vivo assays or therapy.

This is a division of application Ser. No. 575,396, filed Jan. 30, 1984now U.S. Pat. No. 4,707,440.

BACKGROUND OF THE INVENTION

1. Field of the Invention:

The present invention relates to the preparation and use of moleculescarrying attached thereon metal complexing agents or biotin-containingdetectable groups, as well as the products themselves.

2. Description of the Prior Art:

The use of radioactively labelled diagnostic and therapeutic agentsobtained by labeling such agents with metal ions has recently receivedrenewed interest. In this technique, a chelating moiety is covalentlyattached to the molecule of interest, and a radioactive ion is chelatedby the sequestering groups of the chelator. The radioactively labelledagents can then be used both in vitro (for example in radioimmunoassaysystems) and in vivo (for example, both in diagnostic imaging techniquesand in radiation therapy techniques). The use of metal labelling of thenonradioactive type is also of interest, as for example, in theutilization of nuclear magnetic resonance, electron spin resonance,catalytic techniques, and the like.

Different metal chelating groups have been attached to biopolymers inthe prior art. Activated analogues of ethylenediaminetetraacetic acid(EDTA) derived from 1-(p-benzenediazonium)EDTA (I) have been used onproteins: ##STR3##

(See, for example, Meares et al. U.S. Pat. No. 4,043,998, Sundberg etal. Journal of Medicinal Chemistry 17:1304-1307 (1974); or Sundberg etal., Nature 250:587-588 (1974).) The p-benzenediazonium EDTA of formulaI is coupled via an azo linkage to selected tyrosine, histidine or amineresidues of proteins, the latter forming triazines which are acidlabile.

Diethylenetriaminepentaacetic acid (DTPA) is a metal chelator which hasalso been attached to polypeptides (see, for example, Krejcarek et alBiochemical Biophysical Research Communications 77:582-585 (1977),Hnatovich Science 220:613-615 (1983), or Khaw, ibid, 209:295-297(1980).) The chelator is attached through one of its carboxyl groups viaan amide linkage to a protein-derived amino group, as shown in formulaII: ##STR4##

This DTPA conjugate is achieved by first preparing the di-anhydride andreacting the same with a protein. (See for example, Scheinberg, Science215:1511-1513 (1982).) Involvement of the di-anhydride, however, maycause potential crosslinking problems which are either intramolecular orintermolecular. Also, attachment of the chelator through one of itscarboxy groups may remove this carboxy group from consideration as acomplexing moiety, thus decreasing the chelating efficiency, by amodification of the binding affinity constant and geometry.

Wieder et al U.S. Pat. No. 4,352,751 also suggest the attachement ofmetal chelating groups to proteins, utilizingtrans-diaminocyclohexanetetraaacetic acid (DCTA), attached through oneof its carboxy groups to the amino group of a protein. As a model,Wieder et al show the reaction with ethylamine to form compound (III):##STR5##

This compound may suffer from the same problems as the DTPA complex, inthat conjugation occurs through one of the carboxy groups, thuspotentially decreasing the binding affinity, and modifying the geometryof the resulting metal complexes.

Other metal chelating groups have also been attached to biopolymers,e.g., methylpicolinimidate on lysozyme (Benisek et al, J. Biol. Chem.,243:4267-4271 (1968)), ferritin on monoclonal antibodies (Block et al,Nature 301:342-344 (1983)), and the like.

A possible means of overcoming the aforementioned problems of loss ofaffinity, limitation on protein reactive residues, and change ingeometry or crosslinking is disclosed in commonly assigned copendingpatent application Ser. No. 391,440 filed on June 23, 1982 for "ModifiedNucleotides, Methods of Preparing and Utilizing, and CompositionsContaining the Same" by Engelhardt et al, which is herein fullyincorporated by reference. The Engelhardt et al application disclosesthe coupling of a thiocyanate derivative of DCTA to anallylamine-modified deoxyUTP and its possible incorporation intopolynucleotides. See IV: ##STR6##

The use of the deoxyUTP allylamine and its attachment to otherdetectable groups, such as biotin, has also been disclosed (See, forexample Langer et al Proc. Nat. Acad. Sci. 78:6633-06637 (1981)) orco-pending U.S. application Ser. No. 255,223 filed Apr. 17, 1981 at theU.S. Patent and Trademark Office to Ward et al, entitled "ModifiedNucleotides and Methods of Preparing and Using Same," hereinincorporated by reference).

There would be an advantage to utilize the DCTA chelating agent or otherchelating agents without having to extensively modify nucleotides apriori, to utilize physiological chemical process conditions, and toprovide a wide range of alternative methods utilizable in polypeptide,polynucleotide, polysaccharide and small molecule chemistry.

The development of such methodology would allow the use of highaffinity, versatile metal chelating agents such as DCTA, and might alsobe extended and applied to the attachment of other chelators ordetectable moieties, such as biotin.

SUMMARY OF THE INVENTION

The present invention is partly based on the discovery of methods forthe quick, mild and versatile attachment of metal chelating groups andbiotin to polymers, and especially biopolymers such as polynucleotides,polypeptides or polysaccharides. The attachment methods include both theuse of known intermediate linking agents (which, however, had heretoforenot been used for this purpose), or in some instances, includes thedevelopment of novel linking or bridging groups. The invention alsorelates to the products obtained from these methods and extends toproducts comprising both polymers linked to chelators and analoguesthereof, to biotin and analogues thereof, and to various intermediates.

In addition, the invention also provides certain low molecular weight(MW less than about 2,000) molecules linked to a variety of detectableagents such as various chelating agents, and also to biotin moieties.The low molecular weight compounds can be linked by direct bonds or byany known linking arms to any chelating molecule or potentiallychelating molecule. The low molecular weight conjugates between lowmolecular weight compounds and chelating molecules thus have the formula(V):

    A.sup.1 . . . Det.sup.a                                    (V)

where A¹ is a low molecular weight compound of molecular weightpreferably below 2,000, and Det^(a) is biotin or a detectable chemicalmoiety comprising a substituted or unsubstituted metal chelator or acompound capable of yielding a metal chelating compound, most preferablyone of the formula (VI): ##STR7## where M and R³ are defined below; andthe link ". . ." indicates a direct covalent bond or an appropriatespacer arm which does not interfere with the signalling ability ofDet^(a), with the molecular recognition properties of A¹ and whichassures a stable conjugate between A¹ and Det^(a).

In a preferred embodiment, another aspect of the invention comprises adetectable molecule of the formula (VII):

    A.sup.3 (X--R.sup.1 --E--Det.sup.b).sub.m                  (VII)

where

A³ is A² or a polymer, both A² or the polymer having at least onemodifiable reactive group selected from the group consisting of amino,hydroxy, cis 1,2-di OH, halide, aryl, imidazoyl, carbonyl, carboxy,thiol or a residue comprising an activated carbon;

A² is a chemical entity having a molecular weight of less than about2,000;

--X-- is selected from the group consisting of --NH--CO--, --NH--CNH--,--N═N--, --NH--SO₂ --, --OSO₂ --, --NH--N═N--, --NH--CH₂ --, --CH₂--NH--, --O--CO--, --NH--CO--CH₂ --S--, --NH--CO--CH₂ --NH--,--O--CO--CH₂ --, --S--CH₂ --; --O--CO--NH--

--R¹ -- is ##STR8## or a C₁ -C₁₀ branched or unbranched alkyl oraralkyl, which may be substituted by OH;

--Y-- is a direct bond to --S--, or --Y-- is --S--R² --, where R² is aC₁ -C₁₀ branched or unbranched alkyl;

Z_(a) is chlorine, bromine or iodine;

E is O, --N-- or an acyclic divalent sulfur atom;

Det^(b) is a detectable chemical moiety comprising biotin or asubstituted or unsubstituted metal chelator, or a compound capable ofyielding a metal chelating compound, preferably a compound of theformula: ##STR9## where R³ is C₁ -C₄ alkyl or is --CH₂ --COOM, and eachM is a suitable cation;

m is an integer from one to the total number of modified reactive groupson A³.

Yet another aspect of the present invention comprises a detectablemodecule of the formula: ##STR10## wherein A³ is as defined above, j isan electron withdrawing group, K is a signal generating entity or asolid matrix, r is an integer from one to about two and n is as definedabove.

Other specific aspects of the invention comprise individual nucleotides,saccharides or amino acids modified with a group X--R¹ --E--Det asabove. Still other aspects of the invention relate to synthetic methodsof preparing, as well as general methods of using the aforementionedproducts.

The resulting covalent conjugates between the biopolymers or smallmolecules and metal chelators or biotin moieties are utilizable in awide range of applications. For example, the products can be used asdetectable products, by chelating radiometals thereto. They can then beused in a wide range of in vivo and in vitro therapeutic, diagnostic,imaging and assay (immunoassay or hybridization assay) techniques.Biotin labelled biopolymers or small molecules can be used as detectablemolecules wherever biotin/avidin or biotin/streptavidin-based pairs ordetection systems have been used in the prior art. The syntheticpolymers of the invention can be utilized in the same applications asthe biopolymers or small molecules, by attaching the synthetic polymerto biopolymers or small molecules. Thus, for example, such syntheticpolymers can provide numerous radiometals per biopolymer or smallmolecule, which results in a very strong signal being produced.

The ease on introduction, physiological process conditions, versatilityand other such advantages using the DCTA-based chelating agents areparticularly capable of providing a chelator with high affinity, withoutloss of its geometry, and avoidance of crosslinking in its introduction.The methods also have the ability of introducing, at least with certainlinking procedures described hereinbelow, quantitatively more labelingagent per molecule than the prior art.

BRIEF DESCRIPTION OF THE PREFERRED EMBODIMENTS Products

By the small molecular weight entity A² is meant to include the socalled ligands generally involved in immunoassays for theirdetermination. These include drugs which are used for therapeuticpurposes, naturally occurring physiological compounds, metabolites,pesticides, pollutants, enzyme substrates, the reaction product of anenzyme and its substrate, and the like. (For a list of useful entitiesA² see, for example columns 12, 13, 14 and 15 of Rowley et al. U.S. Pat.No. 4,220,722, herein fully incorporated by reference.) For example,included in A² are alkaloids, steroids, lactams, aminoalkylbenzenes,benzheterocyclics, purines, vitamins, prostaglandins, antibiotics, aminoacids, pesticides, and the like. The molecular weight of A² is less thanabout 2,000, especially less than about 1,000.

By the small molecular weight compound A¹, on the other hand areincluded all of the aforementioned compounds for A² with the provisothat A¹ is not a monosaccharide, or a mononucleotide. Preferably A¹ isnot an amino acid either. A¹ thus generally comprises such compounds aspesticides, drugs, pollutants, other physiological compounds, and thelike. For example A¹ includes alkaloids, steroids, lactams,aminoalkylbenzenes, benzheterocyclics, prostaglandins, antibiotics andthe like.

Certain other products within the present invention are detectablepolymers which comprise synthetic polymers and biopolymers such aspolynucleotides, polypeptides or polysaccharides, or larger fractionscontaining these.

By "polynucleotide" is meant to include both polyribonucleotides,polydeoxyribonucleotides, or anypolypurine, poly-pyrimidine or analogue,or combinations thereof. Examples are DNA, RNA, or fragments thereof.

By "polypeptide" is meant to include any polyamino acid chains, whetherhigh or low in molecular weight. These include proteins, hormones,enzymes, immunoglobulins, such as for example, monoclonal antibodies,protein complexes, and the like.

By "polysaccharide" is meant to include any polysaccharide eithernaturally or non-naturally occurring, linear, non-linear or crosslinked,aqueous- soluble or insoluble, unsubstituted or partly or whollysubstituted. These include cellulose, starch, amylose, amylopectin, andthe like.

By "synthetic" polymer is meant to include any synthetic polymer havingat least one modifiable reactive group selected from the groupconsisting of amino, hydroxy, 1,2- cis di OH, halides, aryl, imidazoyl,carbonyl, carboxy, thiol or a residue comprising an activated carbon.Nonlimiting examples of suitable polymers that can be modified to havesuch a modifiable reactive group include polyethylene, polyacrylamide,polyurethane, polystyrene, polyethylene glycol, polybutadiene, polyvinylalcohols and halides and copolymers thereof. If the polymer does notcontain the modifiable reactive group, then such group can be attachedto the polymers by any of the methods well known to those havingordinary skill in the art of organic chemistry.

It is necessary that the entity A³ (which can be either A², supra, or apolymer) prior to reaction have at least one and up to severalmodifiable reactive groups selected from the group consisting of aminogroups (such as for example -amino group of lysine, amino groups inproteins, amino groups in aminopolysaccharides or reactive amino groupson nucleotide bases), hydroxy groups or cis OH groups (such as forexample those in steroids, saccharides, serine or in sugar moieties ofpolynucleotides, such as terminal 3' or 5' hydroxy), carboxyl groups(such as for example aspartate, glutamate, or derivatives thereof),thiol groups (such as for example cysteine), carbonyl groups (such asthose existing in certain steroids, alkaloids, on terminal portions ofnaturally occurring proteins, or obtainable by modification, as is shownhereinbelow), or residues comprising activated carbon groups (such asthe C-3 or C-5 carbon site on tyrosine residues, the C-4 site inhistidine residues, the reactive carbon site on guanine, inosine,cytidine or analogues thereof. For example, guanine has a reactivecarbon atom at position C-8.) Also, A³ can have modifiable reactivegroups such as imidazoyl groups in proteins as part of a histidineresidue or aryl groups as part of tyrosine residue, or halides as partof a synthetic polymer. The reactive carbon atoms of these molecules ormolecular portions of A³, should be capable of convalently reacting withelectrophiles such as diazoaryl functionalities, and undergo coupling(e.g., diazo coupling reactions.)

The modifiable reactive group on A³ may also be present by modificationof A³, and introduction thereinto of such a group. It may also bepresent, for example, in an enzyme cofactor which may be linked,covalently or noncovalently with a polypeptide.

The number of modifiable reactive groups on A³ will depend on thepresence or absence of such groups in A² or certain reactive aminoacids, bases or saccharides in the polypeptide, polynucleotide orpolysaccharide, respectively. This, in the case of the biopolymer, willdepend on the actual chemical composition of the biopolymer, on themolecular weight thereof, as well as the three dimensional structure ofthe biopolymer, and thus the relative accessibility of reactive groupsto the approach and covalent interaction with reactive partners. It isknown, for example, that in proteins there are certain residues whichare more reactive than others, given the fact that they may be closer tothe surface, present in certain active regions, or the like. When thebiopolymer is modified according to the present invention, with anexcess of modifying reagent, the aforementioned factors will determinethe amount and extent of modification. Thus, one, several and possiblyall reactive residues, bases or sugar moieties may react with anappropriate reactive partner.

Alternatively, an individual unit of a biopolymer, such as an individualamino acid or an individual nucleotide or saccharide might be previouslymodified, and then incorporated into a final, build-up biopolymer.

In any event, it is a matter of routine to those of ordinary skill inthe art to estimate whether there exist reactive residues in a givenentity A² or biopolymer, and how many such residues have reacted, inorder to determine the final stoichiometry of the conjugate between A²or the biopolymer and the modifying group. Such techniques asradiolabeling can be used to estimate the extent of modification, and toactually count the number of modified reactive groups. In mostinstances, the number of actually modified groups will be less than thenumber of potentially available modifiable groups of any particularchemical species.

Among the preferred products of the invention are those of the formula(VII):

    A.sup.3 --(X--R.sup.1 --S--Det.sup.b).sub.m                (VII)

where A³ is A² or the biopolymer comprising a polynucleotide,polypeptide or polysaccharide.

--X-- generally comprises a covalent bonding function between one of theA³ -modifiable reactive groups and the group R¹. X may be a singlefunction, such as an amide or ester, or --X-- may be a bridge or linkbetween a modifiable reactive group on A³ and the R¹ group. For example,when X is --NH--CO--, the --NH portion thereof is normally derived froman amine functionality of A¹ or of the biopolymer, and X is a standardamide group. When X is --N═N-- (azo linkage), this linkage is usuallyattached to an activated carbon-containing modifiable group on A² or onthe biopolymer.

R¹ may be unsubstituted phenyl or phenyl substituted by a halogen suchas chlorine or bromine. R¹ may also be a phenyl substituted by a group--Y-- where Y may be a direct covalent bond to --S-- or may be --S--R²--. R² may be divalent C₁ -C₁₀ branched or unbranched alkyl, preferablylower alkyl (C₁ -C₆), most preferably methyl, ethyl, propyl, isopropyl,n-butyl, isobutyl or pentyl.

R¹ may also be a divalent C₁ -C₁₀ branched or unbranched alkyl, asdescribed for R², supra, and preferably lower alkyl (C₁ -C₆), mostpreferably C₂ -C₄. R¹ may also be a C₁ -C₁₀ aralkyl, such as phenylsubstituted by lower alkyl, especially benzyl.

Det^(b) is a detectable chemical moiety which comprises either biotin,or a modified biotin molecule, or comprises Det^(a), which is a metalchelating compound or a compound capable of yielding a metal chelatingcompound. Preferred among these compounds are such molecules as EDTA,DTPA or DCTA or analogues or homologues thereof. Most preferred is thecompound of the formula (VIII): ##STR11##

This formula depicts a cyclohexane-based metal chelator which may beattached to sulfur S through positions 4 or 5, and which carries from 1to 4 metal or nonmetal cations, monovalent cations or the alkaline earthmetals. Thus, with metals of oxidation state +1, each individualcyclohexane-based molecule may carry up to 4 metal cations (where bothR³ groups are CH₂ COOM). As is more likely, with higher oxidationstates, the number of metals will decrease to 2 or even 1 percyclohexane skeleton. The cyclohexane functionality admits of varyingstereochemistry, and the aforementioned formula is not intended to limitthe molecule to any specific stereochemistry. In particular, both aminofunctionalities may be either cis or trans to each other.

The cyclohexane may be unsubstituted (except for the two nitrogenfunctionalities and the sulfur substituent) or may be substituted,especially at the 4-position, with a hydroxy or acylated hydroxy group,such as with a lower acyl substitution.

For purposes of this invention, other cyclohexanebased analogues such asalkyl derivatives (e.g., lower alkyl) or substitution products, whereinthe derivatization or substitution do not interfere with the linking ofthe cyclohexane skeleton to sulfur, with the chelating ability(affinity, geometry, etc.) of the individual chelating moieties, or withthe overall biological activity of the modified A³ are equivalent tothose actually shown. Substitutions which are equivalent for thepurposes of this invention are such as hydroxy, acyl, halogen, amino,and the like.

The A³ moieties having attached cyclohexane moieties may be in the acidform (M=H) or a non-radioactive metal or non-metal form (e.g., M=Mg⁺²,Na' K⁺, Li⁺, NH₄ ⁺, etc.) or in a radioactive metal form.

Any metal capable of being detected in a diagnostic procedure in vivo orin vitro, or capable of effecting therapeutic action in vivo or in vitrocan be used. Both nonradioactive and radioactive metals can be utilizedfor this purpose. Thus, metals capable of catalyzing chemical reactions,metals capable of effecting NMR or ESR spectra, or metals capable ofemitting radiation of various types or intensities could be utilized.Particularly, any radioactive metal ion capable of producing atherapeutic or diagnositic result in a human or animal body or in an invitro diagnostic assay may be used in the practice of the presentinvention. Suitable ions include the following: Antimony-124,Antimony-125, Arsenic-74, Barium-103, Barium-140, Beryllium-7,Bismuth-206, Bismuth-207, Cadmium-109, Cadmium-115m, Calcium-45,Cerium-139, Cerium-141, Cerium-144, Cesium-137, Chromium-51, Cobalt-56,Cobalt-57, Cobalt-58, Cobalt-60, Erbium-169, Europium-152,Gadolinium-153, Gold-195, Gold-199, Hafnium-175, Hafnium-175+181, Indium111, Iridium-192, Iron-55, Iron-59, Krypton-85, Lead-210, Manganese-54,Mercury-197, Mercury-203, Molybdenum-99, Neodymium-147, Neptunium-237,Nickel-63, Niobium-95, Osmium-185+191, Palladium-103, Platinum-195m,Praseodynium-143, Promethium-147, Protactinium-233, Radium-226,Rhenium-186, Rubidium-86, Ruthenium-103, Ruthenium-106, Scandium-44,Scandium-46, Selenium-75, Silver-110m, Silver-111, Sodium-22,Strontium-85, Strontium-89, Strontium-90, Sulfur-35, Tantallum-182,Tecnetium-99m, Tellurium-125, Tellurium-132, Turbium-160, Thallium-204,Thorium-228, Thorium-232, Thallium-170, Tin-113, Titanium-44,Tungsten-185, Vanadium-48, Vanadium-49, Ytterbium-169, Yttrium-88,Yttrium-90, Yttrium-91Zinc-65, and Zirconium-95.

Preferred subgroups within the above formula (VII) are:

--NH--CO-- combined with C₁ -C₁₀ branched or unbranched alkyl;

activated carbon on tyrosine, histidine or guanine, inosine or cytidinecombined with --N═N--Aryl--, where aryl is as defined in formula (VII).

--NH--CO--CH₂ --S-- combined with C₁ -C₁₀ branched or unbranched alkylor with aryl, where aryl is as defined in formula (VII);

Specific examples of modified A³ entities according to the presentinvention are shown in Table I below:

                                      TABLE 1                                     __________________________________________________________________________    A.sup.2                                                                       (modified reac-                                                               tive group)                                                                            X            R           S Det                                       __________________________________________________________________________    Dextran (OH)                                                                           OCN          CH.sub.2CH.sub.2                                                                          S DCTA                                      protein (NH.sub.2)                                                                      ##STR12##   CH.sub.2CH.sub.2                                                                          S DCTA                                      polynucleotide (G,C.sup.8)                                                             NN                                                                                          ##STR13##  S CH.sub.2 CH.sub.2 NHCO(CH.sub.2).sub.4                                         Biotin                                   protein (NH.sub.2)                                                                      ##STR14##   CH.sub.2CH.sub.2                                                                          S DCTA                                      protein (tyr, his)                                                                     NN                                                                                          ##STR15##  S DCTA                                      protein (tyr, his)                                                                     NN                                                                                          ##STR16##  S DCTA                                      polynucleotide (G,C.sup.8)                                                             NN                                                                                          ##STR17##  S DCTA                                      polynucleotide (uridineally amine)                                                      ##STR18##   CH.sub.2CH.sub.2                                                                          S DCTA                                      __________________________________________________________________________

Specific examples of A¹ or A² low molecular weight entities are digoxin,morphine, codein, heroin, diterpene alkaloids, estrogens, DES,barbiturates, amphetamines, catecholamines, chlorpromazine, azepines,diazepines, caffeine, theopylline, cannabinol, THC, penicillins,ethambuzol, chloromycetin, nitrofurantoin, methadone, serotonin,antihistamines, polyhalgenated biphenyls, phosphate esters,thiophosphates, carbamates, and metabolites, derivatives and analoguesthereof.

Other preferred products of the invention are those of the formula (IX):##STR19## wherein a A⁴ is A² or a polymer, both A² or the polymer havingat least one modifiable reactive group selected from the groupconsisting of amino, aryl, imidazoyl and a residue comprising anactivated carbon; A² is a chemical entity having a molecular weight lessthan about 2,000; j is an electron withdrawing group, K is a signalgenerating entity or a solid matrix, n is an integer from one to abouttwo, preferably two, and m is as defined above.

j can be essentially any electron withdrawing group. Preferably, j isselected from the group consisting of chlorine, fluorine, bromine,sulfone groups and iodine, with chlorine being most preferred.

K can encompass virtually any of the signal generating entities used inthe prior art, and any system to be developed in the future. Itcomprises a moiety which generates a signal itself, eg. a radio label ora moiety which upon further reaction or manipulation will give rise to asignal, eg. an enzyme linked system. Non limiting examples of suitablesignal generating entities are disclosed in co-pending, co-assigned,U.S. Patent application Ser. No. 391,440, filed on June 23, 1982. K canbe attached to the benzene ring by any method known in the prior art.Also, K can be a solid matrix such as cellulose. The diazonium productcan be fixed to cellulose by the method disclosed in Seed, U.S. Pat. No.4,286,964.

The preferred products of formula (IX) are: ##STR20##

The products in formula IX are suprisingly stable and are strongelectrophiles. Such stability and strong electrophilicity permits one toattach the products of formula (IX) to A³ when the modifiable reactivegroup is very inert, such as the reactive carbon at the C-8 position ofguanine. It is believed that such stability and strong electrophilicityis due to the electron withdrawing group or groups on the benzene ring.

Other products within the present invention are individual modifiedmononucleotides (ribo- and deoxyribo-) according to the formula (X):##STR21## where P_(z) is ##STR22## or metal or non-metal salts thereof;Q¹ is H or OH;

BA is a modifiable purine or pyrimidine base, such as guanine, inosine,or cytidine.

Preferred among these products are those wherein BA has the formula(XI): ##STR23##

Still other products within the present invention are variousintermediates which are described further hereinbelow.

METHODS

Reactions involving the preferred cyclohexanebased skeleton can becarried out on DCTA or analogues, homologues, or substitutionderivatives thereof, which are prepared according to any of thefollowing Schemes:

SCHEME I Preparation of Bromo-DCTA ##STR24##

Scheme I shows the reduction of 3,4 dinitro phenol (I-1) to 3,4-diaminocyclohexane (I-2); bromination of 3,4-diaminocyclohexane to form3,4-diaminobromocyclohexane (I-3); and further reaction of this compoundwith a halide-substituted carboxymethyl compound to produce thetetracarboxymethyl derivative thereof yielding the title compound (I-4).Details of these reactions can be found in Engelhardt et al, copendingSer. No. 391,440, filed June 23, 1982.

SCHEME II Preparation of Substituted DCTA (exemplified by 4-hydroxy,5-Bromo DCTA) ##STR25##

Scheme II shows the use of 4-cyclohexene-1,2-dicarboxylic anhydride(II-1) as a starting material. Reaction with alcohol followed byhydrazine yields a dihydrazide (II-3) which, when reacted with nitrateand heated, undergoes re-arrangement to a diurethane (II-5). Treatmentof the diurethane with base leads to a diamine (II-6) which can then becarboxyalkylated to yield 1,2-diamino-4-cyclohexenetetraacetic acid(II-7). This compound can, for example, then be treated withN-bromosuccinimide (NBS) to yield 4-bromo-5-hydroxy DCTA derivative(II-8). Details of these reactions can be found in the accompanyingExamples.

SCHEME III Preparation of Diamino Cyclohexane N,N-(dialkyl, diaceticAcid) Derivatives ##STR26##

Scheme III shows the use of 1,4-cyclohexadiene (III-1) to producedibromo derivative III-2, which can further be reacted with N-alkylsubstituted glycine to yield the title compound (III-3).

In the above Schemes I, II or III, it is of course understood thatdifferent halogens, or even pseudohalogens could be used, since theobject is to substitute the cyclohexane with a leaving group capable ofbeing displaced by a mercapto group, SH. Such a leaving group could bechlorine, bromine, cyano, tosylate, mesylate, and the like.

The intermediates or starting materials used in these Schemes (such asfor example the diester cyclohexene (II-2)), can be used for thepreparation of further substituted cyclohexane skeletons as will bereadily appreciated by one of skill in organic chemistry. Thus, a widevariety of modifications and substitutions can be introduced into thecyclohexane skeleton without affecting the basic chemistry of thechelating groups or of the displaceable leaving group.

The attachment of the (substituted or unsubstituted) cyclohexaneskeleton to A³ is carried out via a basic nucleophilic substitutionreaction between the oxygen, nitrogen or preferably, the sulfur atom ofa thiol-containing compound, and the displaceable group or groups on thecyclohexane. The attachment can take any of three general routes.

First, one can attach the A³ --X--R--SH moiety to the leavinggroup-containing cyclohexane by nucleophilic substitution.

Second, one can attach an A³ moiety containing a reactive group, to apreviously prepared X'--R--S--Det^(b), where X' is a group capable ofreacting with the modifiable reactive group on A³, to yield X.

Third, one can use a combination of both the first and secondapproaches, in that A³ is first reacted with part of the bridging group,which in turn is reacted with a previously modified cyclohexane to givethe final conjugate.

In the second approach (Scheme IV, below), one can prepare a diazo arylmoiety-containing cyclohexane (IV-2, bonded to the cyclohexane viasulfur) and react the same with a protein or a polynucleotide asfollows.

SCHEME IV ##STR27##

In the third approach, for example, one can previously modify A³ byreacting modifiable reactive groups thereon with a haloacyl group, andthen reacting this modified A³ with a modified cyclohexane containing anucleophilic group such as a thiol or amine (Scheme V).

SCHEME V ##STR28##

The preparation of haloacyl A³ 's as in Scheme V is shown, for example,in the book "Chemical Modification of Proteins", by Means and Feeney,Holden-Day, Inc., 1971, and in Rowley et al, U.S. Pat. No. 4,220,722,both of which are herein incorporated by reference.

The "A³ -NH" moiety in Scheme V above can also be modified instead bymeans of a compound containing a diazo aryl group (such as a 3,4,5trichlorobenzenediazonium salt) containing a leaving group. Such acompound is known in the tetrafluoroborate form (Korzeniowsky et al,Journal of Organic Chemistry, 1981, 46:2153-2159). Attachment of thiscompound to a modifiable reactive group A³ modifies the resulting A³ byattaching thereto a displaceable chlorine atom. (Such a scheme would bea modification of Scheme IV, above, obtained by inverting the steps).Generally, the attachment of (other) aryl diazonium functions tobiopolymers is known (see Seed, U.S. Pat. No. 4,286,964, and Meares etal, U.S. Pat. No. 4,043,998).

Other possible A³ modifications, especially for biopolymers, useful toprepare the final products of the present invention comprise thereaction of amino groups with diketene to yield acetoacetyl containingA³ 's, possibly followed by reduction. (Means and Feeney, supra, page80-81). The availability of the ketone group of acetoacetyl is useful inreductive amination reactions, where the cyclohexane chelator carries anucleophilic amine.

Amine-containing biopolymers can be reacted with imido esters inalkaline solution to form imido amides, so-called amidines (Means andFeeney, supra, page 90-91). Reaction occurs at moderately alkaline pH,in aqueous solvent and at room temperature. Appropriately substitutedamidines can be prepared which are then capable of reacting withmodified cyclohexane chelators.

Sulfonyl halides and substituted sulfonyl halides, such as chlorides andfluorides, are known to react with amino, sulfhydro, imidazole, andphenolic hydroxy groups of proteins (Means and Feeney, supra, page 97).Reaction with aliphatic hydroxy groups is somewhat slower. Appropriatelysubstituted sulfonyl halides can be used to introduce displaceablegroups, such as displaceable chlorines, into a biopolymer.

Individual modified mononucleotides can be prepared by applying any ofthe above-described methods to said mononucleotides.

Attachment of Det^(b) to polysaccharides can be carried out e.g. byreacting any cis-diol containing polysaccharide with cyanogen biomideand then reacting the resulting water soluble or insoluble activatedpolysaccharide with an appropriately modified, nucleophilic groupcontaining a cyclohexane chelator a precursor thereof, or biotin. Thesame scheme can be applied to the preparation of low molecular weightcis-diol containing molecules, such as digoxin, for example.

The attachment of a detectable moiety comprising biotin would generallyrequire modification of the biotin side chain by attachment of asulfur-containing nucleophile. An example of such a modification isshown below in Scheme VI:

SCHEME VI ##STR29##

Scheme VI exemplifies the use of 1-amino, 2-mercapto ethane. The Schemealso exemplifies the use of 3,4,5 trichlorobenzenediazonium salt, butother such coupling agents can be utilized. For example, when A³ is abiopolymer, the same can be modified with a suitable halide, and themercapto derivative VI-5 can be reacted therewith to yield the finalproduct.

Generally, the reactions with the cyclohexane chelator or derivativesthereof can be carried out with the molecule in the neutralized form(COOH or COONa or COOalkyl form), or in the presence of stoichiometricamounts of other metals such as magnesium. Preparation of the active,detectable cyclohexane moiety containing radiolabelled metal or metalcapable of being detected by or imaged by nonradioactive methodologies(NMR, ESR, etc.) can be carried out after the final step in the organicsynthesis.

Of particular interest is the preparation of a radiolabelled productprior to the utilization of the agent. A method of preparing aradioactively labelled diagnostic or therapeutic molecule generallycomprises contacting a therapeutic or diagnostic agent comprising amolecularly recognizable portion and a chelating portion capable ofchelating with a radioactive metal ion, with an ion exchange materialhaving the radioactive metal ion bound thereto and having a bindingaffinity for the radioactive metal ion less than the binding affinity ofthe chelating portion for the radioactive metal ion, wherein, prior tothe contact, the chelating portion is unchelated or is chelated with asecond metal ion having a binding affinity with the chelating portionless than the binding affinity of the radioactive metal ion, whereby aradiolabelled therapeutic or diagnostic agent is produced by thecontacting, and then separating the radiolabelled therapeutic ordiagnostic agent from the ion exchange material. The so formedradiolabelled material is then immediately used in an in vitro or invivo diagnostic procedure. Such a method is disclosed in commonlyassigned U.S. Pat. No. 4,707,352 filed on even date herewith by Y.Stavrianopoulos for "METHOD OF RADIOACTIVELY LABELLING DIAGNOSTIC ANDTHERAPEUTIC AGENTS CONTAINING A CHELATING GROUP", herein fullyincorporated by reference.

Among other aspects of the invention are various intermediates used inthe aforementioned synthetic procedures. Thus, the invention alsoincludes a modified compound of the formula (XII): ##STR30## where A³ isas defined above, and contains at least one modifiable reactive groupselected from the group consisting of amine and a residue comprising anactivated carbon;

Z_(b) is chlorine, bromine or iodine; and

m is an integer from 1 to the total number of modified reactive groupson A³.

This modified A³ is useful in preparing the preferred final detectableproducts of the invention.

The invention also includes a compound of the formula (XIII): ##STR31##or the 4-hydroxy or acyloxy derivative thereof, where

M is as defined previously;

--S-- is divalent sulfur atom; and

Q² -- is H; branched or unbranched C₁ -C₁₀ alkyl or aralkyl whichcarries a group selected from the group consisting of --OH, --SH, --NH₂,--CONHNH₂, or --C--Lv, where Lv is a displaceable leaving O group; or Q²is ##STR32## where Z_(c) is hydrogen, chlorine, bromine or iodine, and Jis --NH₂ or --N₂ ⁺ CA⁻, where CA⁻ is a counteranion.

Examples of Lv are --N₃, --Cl, --Br, tosylate, mesylate, and the like.Examples of CA⁻ are fluoroborate, tetrafluoroborate, tosylate,perchlorate, and the like.

Still other intermediates are modified mononucleotides of formula XIV:##STR33## where P_(z), BA and Q¹ are as defined above.

The modified mononucleotides (XIV) can be integrated into apolynucleotide and then reacted with appropriate SH-group containingcyclohexane chelator or biotin. Alternatively, the modifiedmononucleotides (XIV) are reacted with a cyclohexane chelator or biotin,and the resulting products are incorporated into a polynucleotide.

Still other intermediates include compounds of the formula XV: ##STR34##wherein j, n and K are as defined previously, and CA⁻ is a suitablecounteranion.

The preparation of small molecular weight chelator-containing compounds(V):

    A.sup.1. . . Det.sup.a                                     (V)

can be carried out by any of the well known methods of linking metalchelating moieties or potential metal chelating moieties to molecules.For example such chelators as EDTA, DTPA or DCTA can be attached toamino or hydroxy groups of A¹ with formation of amides or esters.Diazoaryl containing chelators or potential chelators can be attached toactivated aromatic groups on A¹.

APPLICATIONS

The uses and applications of the chelator or biotin-containing compoundsof the invention are unlimited, and extend to all of those uses to whichdetectably labelled compounds of this type had been put in the priorart. For example, any compound desired to be detected and analyzed in asample can be modified according to the techniques of the presentinvention. Of particular interest are the modification of antibodies foruse in immunoassay procedures, such as sandwich immunoassay procedures.Also of interest is the modification of drugs for radioimmunoassayprocedures or of proteins associated with or known to be present onmicroorganism walls or membranes. Detectably labelled proteins preparedin such manner can also be used in competitive immunoassay procedures.Labelled polynucleotides can be used in hybridization assays.

Another use of the detectable compounds, especially biopolymers, of theinvention is in imaging, especially with monoclonal antibodies. Thesecan be modified according to the techniques of the invention and allowedto carry a metal onto a given site in a living material, such as ananimal body. Detection can then be carried out by radiologicaltechniques. A metal carried to such site can also be chosen to be anemitter, thus producing localized radiotherapy (see, e.g., Denardo, S.et al, in "Nuclear Medicine and Biology," Vol. IV, Pergamon Press,Paris, France, 1982, pp. 182-185.)

Of particular interest is the detection and identification of viral andbacterial DNA sequences.

Polynucleotide products prepared according to this invention can beutilized to diagnose genetic disorders by preparing a polynucleotidecomplementary to a DNA gene sequence which is associated with a geneticdisorder, and detecting the presence of hybridization events. Detectablepolynucleotides can also be used in chromosomal karyotyping, whichcomprises using a series of modified polynucleotides corresponding to aseries of defined genetic sequences located on chromosomes, and thendetecting hybridization.

Another use includes a method for identifying or locating hormonereceptor sites on the surface of cells, which comprises binding ahormone receptor binding compound which is a detectable biopolymer underthis invention, and then detecting binding by means of a detectionsystem.

Another use of the metal chelating conjugates of the present inventionis in the removal of unnecessary and dangerous accumulations of metalions from targets specified by a particular biopolymer to which thechelator portion is attached. Generally, the chelator-labelled moleculesof the invention can be used in other detection systems known or laterdiscovered. These uses include chelation with nonradioactive heavy metalions for use as radioopaque agents, chelation with a metal iondetectable by NMR, chelation with a metal having catalytic propertiesand capable of catalyizing a chromogenic chemical reaction, and thelike.

Biotin bound compounds can be detected by carrying out their incubationwith avidin or streptavidin, wherein the avidin or streptavidin iscovalently coupled to a detectable label such as an enzyme (e.g.,alkaline phosphatase or peroxidase) capable of catalyzing the formationof a chromogenic product. This is the standard, well known enzyme linkedimmunoassay system (ELISA) and is described in co-pending patentapplication Ser. No. 392,440 filed on June 23, 1982 to "ModifiedNucleotides, Methods of Preparing and Utilizing, and CompositionsContaining the Same" by Engelhardt et al, herein fully incorporated byreference.

The present invention also lends itself readily to the preparation ofkits. A kit may comprise a carrier being compartmentalized to receive inclose confinement therein one or more container means or series ofcontainer means such as test tubes, vials, flasks, bottles, syringes, orthe like. A first of said container means or series of container meansmay contain a detectable compound according to the present invention, orthe compound present in varying concentrations so as to provide thecapability of building a standard interpolation curve. If the chelatorbound compound is provided in nonradiolabelled metal form, anothercontainer means may contain a desired radiometal and appropriate ionexchange materials to provide for the exchange of "cold" for radioactivemetal. Thus, a user can easily label the chelator conjugated compoundwith a radioactive metal. Alternatively, a second container means orseries of container means might contain avidin or streptavidin, suchmolecules covalently bound to an enzyme for use in an enzyme immunoassaysystem.

Having now generally described this invention, the same will be betterunderstood by reference to certain specific examples which are includedherein for purposes of illustration only and are not intended to belimiting unless so specified.

EXAMPLE 1 Synthesis of 1,2 transdiaminocyclohexene tetraacetic acid

Step 1: ##STR35##

4-Cyclohexene-1,2-dicarboxylic anhydride (154 grams, 1.01 moles) wasdissolved in 700 ml of anhydrous 200 grade ethanol. Concentratedsulfuric acid (40 ml) was added, and the reaction mixture was refluxedfor three hours. Approximately 350 ml of ethanol was removed bydistillation. The distillation also removed water as part of theethanol-water azeotropic mixture. Approximately 350 ml of absolute 200grade anhydrous ethanol was added, and then the reaction was allowed toreflux for another 90 minutes. Then, using a rotary evaporator equippedwith an air pump (not an aspirator) approximately 400 ml of solvent wasremoved. One liter of ether was added to the remaining reaction mixture,and the reaction products were dissolved. The reaction mixture waspoured into an ice-water mixture (1 liter water and 1 liter ice) andthen placed in a separatory funnel. After shaking and allowing the etherand aqueous layers to separate, the aqueous phase was removed anddiscarded. The ether phase was extracted three times with 100 mlaliquots of cold 5% sodium bicarbonate to remove any H₂ SO₄ andmonoester. (The pH of the final bicarbonate extract was the same as afresh 5% bicarbonate solution.) The ether layer was dried over anhydrousNa₂ SO₄ with stirring for 90 minutes. The Na₂ SO₄ was removed byfiltration. The ether was removed from the product4-cyclohexane-1,2-dicarboxylate ethyl diester on the rotary evaporatorand air pump. Gradually, once most of the ether had been removed, thewater bath temperature was increased to 90° C. to remove the residualether quantitatively. A yield of 160 grams of liquid diester wasobtained.

Step 2: ##STR36##

4-Cyclohexene-1,2-dicarboxylate ethyl diester (160 g, 0.707 moles) and250 ml of commercial grade hydrazine (54% hydrazine=4.22 moles) wereplaced in a reflux apparatus without stirring. The oil bath was thenheated to 130° C. under argon. (The diester and the hydrazine remainedin two phases.) Absolute (200 grade) ethanol (less than 100 mls) wasadded with rapid stirring through the top of the reflux condenser untilone phase was obtained. (The milky phase disappeared.) The reactionmixture was refluxed at 130° C. under Argon with stirring (to avoidbumping) for 24 hours. The product 4-cyclohexene 1,2-dihydrazineprecipitated as it formed. While the reaction mixture was still warm, itwas poured into a 2-liter beaker and allowed to cool. The mixturesolidified, and the crude product was filtered on a Buchner Funnel.During filtration, the solid, while still on the Buchner Funnel, waswashed with ethanol. The crude hydrazide was recrystallized as follows:

One liter of 85% ethanol (in water) was heated with stirring. A portion(40-50 g) of the crude hydrazide was added. The hydrazide dissolvedwhile the mixture was boiled for 10 minutes. The solution was decantedto remove any insoluble material, and the solution was allowed to coolslowly at room temperature. As the solution cooled, the hydrazideprecipitated. It was filtered using a Buchner Funnel and then washedwith a small amount of ethanol.

The volume of the filtrate was adjusted to 1 liter with absoluteethanol. The filtrate was then heated with stirring, and another 40-50 gof the crude hydrazide was recrystallized as described above. Theprocedure was repeated until all of the crude hydrazide had beenrecrystallized. A total of 120 grams of the hydrazine was obtained.

Step 3: ##STR37##

4-Cyclohexene-1,2-dihydrazide (20 g, 0.101 moles) was dissolved in 200ml of 2N sulfuric acid. Ether (250 ml) was added, and the reaction flaskwas placed in an ice-salt bath and allowed to cool to -2° to -3° C. Overa 5-10 minute period solid sodium nitrite (13.9 g, 0.202 moles) wasadded with vigorous stirring. The temperature of the mixture was notallowed to exceed 5° C. Stirring was continued for 10 minutes. Thereaction mixture was poured into a cold separatory funnel (theseparatory funnel and a one-liter Erlenmeyer flask had been chilled inthe freezer), and the phases were allowed to separate. The ether phasewas transferred to the cold Erlenmeyer. The aqueous phase was extractedtwo times with approximately 100-150 ml portions of cold ether asfollows. The aqueous phase was stirred with the ether in an ice bath (nosalt) for 10 minutes, and then the phases were separated using the coldseparatory funnel. The cold ether extracts were combined and thenextracted two times with 30 ml portions of cold 5% sodium bicarbonatesolution. The ether phase was dried over 60 g of anhydrous sodiumsulfate by stirring for 30 minutes at 4. The sodium sulfate was removedby filtration through glass wool. The product 4-cyclohexene-1,2-diazide(in ether) was not purified or characterized. In order to proceed withthe next reaction, the azide was transferred from ether to benzene asfollows. An aliquot (approximately 1/3 total ether volume) of the ethersolution containing azide was added to approximately 150 ml anhydrousbenzene. The ether was evaporated quickly using the rotary evaporatorand air pump. The temperature of the water bath was kept below 45° C.during removal of the ether. Additional aliquots of the azide in ether(approximately 1/3 each time) were added until all of the azide had beentransferred to the benzene.

Step 4: ##STR38##

100 ml absolute toluene (100 ml) and benzyl alcohol (30 ml, 0.289 moles)were placed in a flask and heated to 70° C. Aliquots (10 ml) of the4-cyclohexene,2-dicarboxyl azide in benzene solution (from the previousstep) were added slowly (total time for approximately 150 mls benzenesolution was approximately 30 minutes) to contain the N₂ evolution, andto prevent the temperature from exceeding 80° C. All of the benzenesolution from the previous step was added.

At this point, another 20 g of hydrazide from the prior step wasconverted to the azide. However, the above procedure was amended asfollows. Only 100 ml (not 150) benzene was used per 20 g of hydrazidestarting material. The remaining toluene-benzyl alcohol mixture fromabove was supplemented with 20 ml benzyl alcohol, and then the 100 mlbenzene solution was reacted in 10 ml aliquots as described above. Thisprocedure was repeated for a third 20 g batch of hydrazide. The threebatches were combined and 10 ml of benzyl alcohol was added (for a totalof 0.772 moles per 60 g hydrazide). As much benzene as possible wasremoved by distillation, and then the remaining reaction mixture wasallowed to reflux overnight at a bath temperature of 120° C. During thistime, the reaction mixture became reddish-brown. The toluene was removedon the rotary evaporator equipped with a vacuum pump (0.5 mm Hg). Aceticacid (100-200 ml 50% glacial acetic acid in water) was added to thereaction flask, and the mixture was stirred with a glass rod. Thedesired urethane product (4-cyclohexene-trans-1,2-dibenzylurethane) wasinsoluble, but the reddish-brown color went into the liquid phase. Theurethane product was filtered on a Buchner funnel and washed with cold50% glacial acetic acid in water. For analysis, the urethane wasrecrystallized from 50% glacial acetic acid in water. A yield of 60grams of the urethane was obtained.

Step 5: ##STR39##

4-Cyclohexene-trans-1,2-dibenzylurethane (60 g) from the previous stepwas added to 300 ml of 7N sodium hydroxide and refluxed for one hour.Using a very efficient condenser, approximately 70 ml water was removedby distillation to give a final sodium hydroxide concentration ofapproximately 10N. At this concentration of sodium hydroxide, the sodiumcarbonate produced during the reaction precipitated. The mixture wasallowed to cool, and then the sodium carbonate was removed by filtrationon a Buchner Funnel. The sodium carbonate was washed with 100-150 mln-butyl alcohol. The resulting filtrate had two phases. The filtrate wasplaced in a separatory funnel and shaken, and the water layer wasremoved. Then 150 ml water and concentrated hydrochloric acid were addeduntil the pH was 1. The mixture was shaken and the phases were allowedto separate. At this point, most of the producttrans-1,2-diamino-4-cyclohexene was in the dihydrochloride form in theaqueous phase. Concentrated hydrochloric acid (50 ml) was added to theorganic phase, and more of the product amine dihydrochlorideprecipitated. The solid was filtered on a Buchner Funnel and then driedin a dessicator over sodium hydroxide pellets to remove the hydrochloricacid. The aqueous phase containing most of the product was evaporated todryness on the rotary evaporator equipped with a vacuum pump (0.5 mmHg). The two batches of the product were combined and used in the nextstep without further purification. It was assumed that there was 100%conversion to the amine.

Step 6: ##STR40##

The amine trans-1,2-diamino-4-cyclohexene in the hydrochloride form(assumed quantitative conversion from urethane, 0.158 moles) and 75 gchloroacetic acid (0.79 moles) (both solids) were combined in anErlenmeyer flask equipped with a large stirrer and an ice bath. Cold 7Nsodium hydroxide (approximately 113 ml) was added slowly to neutralizethe chloroacetic acid. Care was taken to keep the reaction mixture cool(below 20° C.). Addition of sodium hydroxide then produced a suspensionof the amine. The reaction mixture was gently stirred and heated in a55° C. oil bath. Using a pH meter to monitor the pH, 7N sodium hydroxidewas added to keep the pH between 9 and 10. Once all the amine haddissolved, the oil bath temperature was increased to 90°-95° C. for onehour. During this time, the pH was maintained between 9 and 10 with 7Nsodium hydroxide. The reaction mixture was allowed to cool, and then thepH was adjusted to 1.2 with concentrated hydrochloric acid. One liter ofacetone was added slowly at room temperature, and the bulk of the sideproduct, sodium chloride, precipitated. The sodium chloride was removedby filtration on a Buchner Funnel. The desired product,trans-1,2-diamino-4cyclohexenetetraacetic acid, remained in thefiltrate, which was light red-brown in color. The acetone was removed bya rotary evaporation, using a bath temperature at the end of 80° C. Theproduct remained in aqueous solution and was allowed to cool overnight.A slight precipitate appeared. The pH was readjusted to 1.2 (it was0.9), and the walls of the flask were scratched to inducecrystallization. Further stirring with a magnetic stirrer resulted infurther precipitation of the product. The product,trans-1,2-diamino-4-cyclohexenetetraacetic acid (11 g), crystallizedfrom the first crop, and 6 g additional product precipitated slowly overa one month period. The precipitate was collected by filtration, washedwith cold water, and dried under vacuum at 100° C. A total of 17 gramsof the monounsaturated DCTA product was obtained.

EXAMPLE 2 4-bromo-5-hydroxy-DCTA ##STR41##

N-bromosuccinimide (NBS) was recrystallized according to the followingprocedure. One liter of water was placed in a flask equipped with amagnetic stirrer and heated to 75° C. Crude NBS (100 g which had beenground to a fine powder using a mortar and pestle) was added withstirring and then stirred for two minutes. The solution was filteredquickly through a warm Buchner Funnel. (The funnel must be warm;otherwise a precipitate will form during the filtration.) The filtratewas poured quickly into an ice cold beaker to precipitate the NBS whileminimizing its decomposition. The NBS was lyophilized overnight toremove traces of water. A total of 80 grams was recovered.

Trans-1,2-diamino-4-cyclohexenetetraacetic acid (1 g, (0.0028 moles)free acid was dissolved in 1.5 ml 7N NaOH to give a final pH ofapproximately 5.2. The solution was cooled to 10° C. and then added to0.5 g NBS (0.0028 moles) on a glass tube equipped with a magneticstirrer and an ice bath. The reaction mixture was stirred with thethermometer, and the temperature was kept at 8°-10° C. for 1 hour. Themixture was stirred overnight at 4° C. The reaction was judged to becomplete when solid was no longer visible. The product4-bromo-5-hydroxy-DCTA, in the form of the sodium salt, was precipitatedby the addition of 10 volumes of dry methanol. The product was collectedby filtration on a Buchner Funnel, washed with a small amount ofmethanol, and quickly air dried. Exposure to light was minimized toprevent decomposition. The product was placed in an aluminum-foilcoveredflask and lyophilized under high vacuum overnight. A total of 1 gram ofthe sodium salt was obtained. (The stereochemistry around the Br-- andOH-- bearing carbons has not yet been fully elucidated. For purposes ofthis invention, however, the stereochemistry is not critical.)

EXAMPLE 3 5-hydroxy-DCTA-4-β-thiopropionic acid hydrazide ##STR42##

Before this reaction can be performed, the reactant β-mercaptopropionicacid hydrazide must be synthesized, according to the followingprocedure. β-Mercaptopropionic acid (53 g, 0.5 mole, liquid) was mixedwith 0.6 moles NaOH (24 g), 0.2 moles KI (33.2 g), and 0.5 molescrystalline I₂ (126.9 g). The bisthiol formed and precipitated at once.It was filtered on a Buchner Funnel, recrystallized from water, anddried for 15 minutes at 95° C. to give 48 g (0.23 moles, 92% yield) ofthe bisthiol dipropionic acid.

The bisthiol dipropionic acid (48 g, 0.23 moles) was dissolved in 400 mlof absolute ethanol containing 10 ml concentrated sulfuric acid (0.138moles), and the mixture was refluxed for 2 hours. Ethanol (200 ml) wasremoved by distillation. Absolute ethanol (an additional 200 ml) wasadded to the mixture, the mixture was refluxed for another 2 hours, and200-250 ml ethanol were removed by distillation. Ether (500 ml) wasadded to the product solution and then the mixture was poured onto ice.Solid sodium bicarbonate (30.7 g, 0.366 moles) was added with stirringto neutralize the solution. The phases were separated in a separatoryfunnel. The aqueous phase was discarded. The ether phase was washed oncewith 100 ml cold 5% sodium bicarbonate followed by one wash with 200 ml0.1M sodium chloride. The ether layer was dried over 50 g anhydrous Na₂SO₄ with stirring for 90 minutes. The Na₂ SO₄ was removed by filtration.The ether was removed from the product [(CH₃ --CH₂ --O--CO--CH₂ --CH₂S)₂ ] on the rotary evaporator equipped with an air pump. Gradually,once most ether had been removed, the water bath temperature wasincreased to 90° C. to remove the residual ether quantitatively. Thecrude product was then mixed with 100 ml commercial grade hydrazine (54%hydrazine=1.7 moles) and 100 mls absolute ethanol to convert the ethylester to the hydrazide. The mixture was refluxed with stirring overnightunder argon to prevent oxidation. The solution became yellowish brown.The ethanol was removed by rotary evaporation. The solid product wastriturated with 200 ml absolute ethanol and then collected by filtrationon a Buchner Funnel. The product, which was a yellow powder, wasrecrystallized from 250 ml ethanol to yield 20.3 g bisdithiol hydrazide(NH₂ --NH--CO--CH₂ --CH₂ S--)₂ (0.17 moles, 37%). The product wasreduced in one step with a 30-fold molar excess of sodium borohydride asfollows.

Bis dithiohydrazide (0.75 m moles) was dissolved in 1.0 ml water. Solidsodium borohydride (2.25 moles, 85 mg) was added, and the reactiontemperature was maintained at 25° C. with occasional cooling. Formationof the product thiol was monitored at 5 minute intervals as follows. A 5μl aliquot was dissolved in 200 μl cold water, and the remainingborohydride was destroyed by the addition of 50 μl 1N HCl. Thiol wasdetermined with Cleland's reagent.

When the reaction was complete, the mixture was cooled and 1N HCl wasadded, dropwise, to destroy the excess borohydride. HCl (1N) was addeduntil gas evolution was no longer observed. The reaction mixture wasneutralized to pH 7.8 with 5N NaOH. Indicating paper was used to monitorthe pH. At this point, the desired final product, β-mercaptopropionicacid hydraze, was obtained. It was used immediately as follows.

4-Bromo-5-hydroxy-DCTA (441 mg, 1 mmole) and 1 ml 2M potassium carbonatewere added to the β-mercaptopropionic acid hydrazide, reacted 2 hrs. at95° C. under argon and then the reaction mixture was cooled, dilutedwith water and loaded onto a Dowex 1 column. The unreacted hydrazide didnot bind to the column and was monitored in the column break-throughwith picrylsulfonic acid (production of a red colored solution). Thecolumn was washed with 0.1N acetic acid and then the product5-hydroxy-DCTA-4-thiopropionic acid hydraze was eluted with 0.2N HCl.The hydrazide positive fractions (as monitored by reaction withpicrylsulfonic acid) were combined and then neutralized with 5M NaOH. Ayield of 0.6 mmoles was obtained.

EXAMPLE 4 4-aminoethylthio-t-hydroxy-DCTA(5-hydroxy-DCTA-4-β-thioethylamine) ##STR43##

4-Bromo-5-hydroxy-DCTA (882 mg, 2.0 mmol) and 340 mg2-aminoethanethiolhydrochloride (3.0 mmol) were combined in 1.0 ml 2Mpotassium carbonate under argon, and then heated for 2 hours at 90° C.The reaction was monitored by following the disappearance of thiol usingCleland's reagent [5,5'-dithio-bis-(2-nitrobenzoic acid)]. Afterapproximately 90% of the thiol had disappeared, the reaction mixture wasdiluted approximately 50-fold with oxygen-free water and then loadedonto a 6 ml Dowex-1 column (acetate form, 0.8 meg/ml resin). Theunreacted 2-aminoethanethiol hydrochloride did not bind the column. Thecolumn was washed with 0.1N acetic acid. The wash was monitored for thepresence of amine using ninhydrin. Some amine bound as salt to theproduct was washed off as peak and then levels of amine plateaued. Atthis point, the column was eluted with 0.2M HCL (approximately pH 1).During the elution, part of the product 4-aminoethylthio-5-hydroxy-DCTAprecipitated; however, when the pH reached approximately 1, the productdissolved, so care must be taken to elute all of the product.

The eluant was tested for amine using ninhydrin. All amine-positivefractions were combined and lyophilized. A yield of 1.07 g of theproduct as the amine trihydrochloride was obtained.

EXAMPLE 5 Synthesis of Modified Nucleotide--dUTP allyamine

(a) Preparation of mercurated dUTP

Deoxyribouridine triphosphate (dUTP; 554 mg) was dissolved in 100 ml of0.1M sodium acetate buffer pH 6.0, and mercuric acetate (1.59 gm, 5.0mmoles) was added. The solution was heated at 50° C. for 4 hours, thencooled on ice. Lithium chloride (392 mg, 9.0 mmoles) was added, and thesolution was extracted six times with an equal volume of ethyl acetateto remove excess HgCl₂. The efficiency of the extraction process wasmonitored by estimating the mercuric ion concentration in the organiclayer using 4,4'-bis (dimethylamino)-thiobenzophenone (A. N.Christopher, Analyst, 94, 392 (1969)). The extent of nucleotidemercuration, determined spectrophotometrically followed iodination of analiquot of the aqueous solution as described by Dale et al. (R. M. K.Dale, D. C. Ward, D. C. Livington, and E Martin, Nucleic Acid Res. 2,915 (1975)), was routinely between 90 and 100%. The nucleotide productsin the aqueous layer, which often became cloudy during the ethyl acetateextraction, were precipitated by the addition of three volumes ofice-cold ethanol and collected by centrifugation. The precipitate waswashed twice with cold absolute ethanol, once with ethyl ether, and thenair dried. These thus-prepared mercurated nucleotides were used for thesynthesis of the allylamine-nucleotides without further purification.

(b) dUTP allylamine

The mercurated nucleotide (of step a) was dissolved in 0.1M sodiumacetate buffer at pH 5.0 and adjusted to a concentration of 20 mM (100OD/ml at 267 nm). A fresh 2.0M solution of allylamine acetate in aqueousacetic acid was prepared by slowly adding 1.5 ml of allylamine (13.3mmoles) to 8.5 ml of ice-cold 4M acetic acid. Three ml (6.0 mmoles) ofthe neutralized allylamine stock was added to 25 ml (0.5 mmole) ofnucleotide solution. One nucleotide equivalent of K₂ PdCl₄ (163 mg, 0.5mmole) dissolved in 4 ml of water, was then added to initiate thereaction. Upon addition of the palladium salt (Alfa-Venton) the solutiongradually turned black with metal (Hg and Pd) deposits appearing on thewalls of the reaction vessel. After standing at room temperature for18-24 hours, the reaction mixture was passed through a 0.45 mm membranefilter (nalgene) to remove most of the remaining metal precipitate. Theyellow filtrate was diluted five-fold and applied to a 100 ml column ofDEAE-Sephadex TM A-25 (Pharmacia). After washing with one column volumeof 0.1M sodium acetate buffer at pH 5.0, the products were eluted usinga one liter linear gradient (0.1-0.6M) of either sodium acetate at pHapproximately 8-9 or triethylammonium bicarbonate (TEAB) at pH 7.5. Thedesired product was in the major UV-absorbing portion which elutedbetween 0.30 and 0.35M salt. Spectral analysis showed that this peakcontained several products. Final purification was achieved by reversephase--HPLC chromatography on columns of Partisil--ODS2, using either0.5M NH₄ H₂ PO₄ buffer at pH 3.3 (analytical separations) or 0.5Mtriethylammonium acetate at pH 4.3 (preparation separations) as eluents.The 5'-triphosphates of 5-(3-aminopropen-1-yl) uridine (the allylamineadduct to uridine) were the last portions to be eluted from the HPLCcolumn, and they were clearly resolved from three as yet uncharacterizedcontaminants.

EXAMPLE 6 dUTP allylamine labelled with 5-hydroxy-DCTA-4-β-thiopropionicacid hydrazide

5-Hydroxy-DCTA-4-β-thiopropionic acid hydrazide (12 mmoles, 4-foldexcess) was dissolved in 0.3 ml water. HCl (50 l, 1N) was added, and thereaction mixture was cooled to 0° C. Cold 0.05M NaNO₂ (0.3 ml) was addedto the reaction mixture which was allowed to react at 0° C. for 10minutes. At this point, the DCTA hydrazide had been converted to theazide (--N₃). Potassium carbonate (25 μl 2M) was added to neutralize thereaction mixture and 10 μl 10M magnesium sulfate was added to neutralizethe charge. The pH was adjusted to approximately 8 with 15 μl 2Mpotassium carbonate and then 500 μl of dUTP allylamine (3 mmoles in 0.6MNaCl) and 100 μl cold 5% sodium bicarbonate were added at 0° C. Themixture was allowed to react overnight (approximately 12 hours) at 0° C.The product was separated from unreacted starting materials as follows.The reaction mixture was neutralized to pH 7 with 1N HCl and then loadedonto a 1 ml hydroxylapatite column which had been equilibrated with0.01M potassium phosphate (equimolar in monobasic and dibasic potassiumphosphate, approximate pH 6.8). 0.3 ml fractions were collected. Thecolumn was washed with the same buffer (4 ml) until no more absorbanceat A₂₆₀ or A₂₅₅ eluted.

The product DCTA-labelled dUTP and any unreacted dUTP allylamine wereeluted with 0.3M potassium phosphate buffer (equimolar in monobasic anddibasic potassium phosphate). All fractions with an absorbance of 290 nmwere pooled for a total of 0.9 ml. Total A₂₉₀ =17.8 OD: E_(dUPT) AA290=8 nm⁻¹, yield of 2.3 μmoles=77%.

The product was transferred to a different buffer according to thefollowing procedure. The product (0.9 mls) was diluted 20-fold with coldwater and then loaded onto a 0.5 ml DE-52 cellulose column (chlorideform). All A₂₉₀ was absorbed. The column was washed with 0.5 ml (one bedvolume) of the 0.05M NaCl. The product was eluted with 0.6M NaCl in avolume of 1.0-ml. A₂₉₀ recovered=17.8 OD, for 100% recovery.

A control reaction using dTTP instead of dUTP allylamine was run inparallel and processed similarly. Ten μl of each were diluted with 20 μlglycine 0.05M ammonium acetate in the presence of radioactive nickel andallowed to bind for 15 minutes. Each batch was loaded onto a Dowex 50(NH₄ ⁺ form) column. Only the product of the reaction with dUTPallylamine was labelled with the radioactive nickel. At least 84% of theproduct was the DCTA analogue.

The structure of the chelator-labelled dUTP allylamine product was asfollows: ##STR44##

EXAMPLE 7 Protein (Immunoglubulin G) labelled with 5-hydroxy-DCTA

One equivalent DCTA-hydrazide (in water) was treated with a 10-foldexcess of 1N HCl and cooled to 0° C. One equivalent of cold 0.05M NaNO₂solution in a precooled pipette was added to the reaction mixture at 0°C. The mixture was allowed to react at 0° C. for 10-15 minutes. Thereaction mixture was neutralized (to approximately pH 8.5) with cold 5%sodium bicarbonate. This also resulted in high salt molarity. TheImmunoglobulin G protein was added and incubated overnight at 0° C. Theexcess activated DCTA was retained by a G-50 column, and the proteincame through at the void volume.

EXAMPLE 8 DCTA-SH ##STR45##

One millimole of DCTA-bromide was added to 5 ml of 50% DMF containing0.2 ml of 1,2-dithioethylene and 0.5 ml of triethylamine. The mixturewas incubated under argon for 2 hours at 60°-70° C. After reaction themixture was diluted with oxygen-free H₂ O to 50 ml, the pH was adjustedto 4.0-4.5 with glacial acetic acid and the excess HS--CH₂ --CH₂ --SHwas extracted three times with 15 ml benzene by stirring (not shaking).The water phase was then loaded onto a Dowex AG-1 column, 9 ml bedvolume. The column was washed with 50 ml of 0.1M acetic acid solutionuntil the flowthrough was thiol free. The DCTA-SH was then eluted with0.25M HCl. The thiol containing fractions were combined, evaporated todryness under reduced pressure at 40° C. and the free acid (300 mg) wasstored at -20° C. under argon.

EXAMPLE 9 Activation of DNA with 3,4,5-Trichloroaniline

100 mg of 3,4,5-trichloroaniline was dissolved in 2.5 ml of 0.5M HCl in50% DMSO and cooled on ice, under vigorous stirring, an equimolar amountof NaNO₂ from a cold 1M solution was added, as rapidly as possible, andthen stirring was continued for 10 minutes. 1 mg of 3H or fd DNA in 300ml of water were mixed with 300 μl 2M cacodylate buffer pH 6.6 and 600μl DMSO. (By addition of DMSO the pH of the solution rises to 8.3). 20μl of the freshly prepared diazonium solution were added thereto and themixture was incubated for two hours at room temperature. The slightprecipitate which appeared during the incubation was removed bycentrifugation. The solution was then made 0.4M with ammonium acetateand the DNA was precipitated with ethanol.

EXAMPLE 10 Reaction of Trichloroaniline-activated DNA with thiols.Example of Reaction with DCTA-SH

fd DNA activated with 3,4,5-Trichloroaniline (Example 9) was dissolvedin 0.1M sodium hydroxide with an equal amount of 0.1M K₂ HPO₄. Thissolution was treated with an equal volume of 0.1M DCTA-SH (Example 8)and incubated under argon at 65° C. for 2 hours. The precipitateddisulfides were removed by centrifugation and the DNA was purified byG50 chromatography and stored at -20° C. Using radioactive Ni toestimate the level of derivatized DNA, it was determined that 60% ofguanines had been labelled.

EXAMPLE 11 Biotin-SH

Three millimoles of Biotin-NHS ester were dissolved in 25 ml ofanhydrous DMF and mixed with a 1M solution of cysteamine hydrochloridein 12 ml of 0.5M sodium bicarbonate and the mixture was incubated atroom temperature overnight. During the incubation, a heavy precipitateappeared. The liquid was removed under reduced pressure at 45° C. andthe residue was suspended in 50 ml absolute ethanol, 1 g of NaBH₄ wasadded and the suspension was stirred for one hour at 75° C. The ethanolwas removed, cold 1M HCl was added to bring the pH to 4.5, and the waterwas removed under reduced pressure at 35° C. (All these operations wereperformed under an argon atmosphere to prevent oxidation of the thiol.).The solid residue was powdered and triturated with 4 ml of colddeareated 0.01M acetic acid. This procedure was repeated twice and theresidue was lyophilized. TLC chromatography showed that the main biotinspot contained thiol; two minor spots were thiol negative. In allreactions, the amount of biotin used was based on the thiol content. Itis possible to use Biotin-SH in the reaction withtrichloroaniline-activated DNA, in analogy to Example 10.

EXAMPLE 12 Labelling of the 3,4,5-trichloroaniline DNA with DCTA-SH

0.5 mg of the activated DNA (Example 9) in 0.2 ml of water were mixedwith 2.0 ml of 0.5M triethylammonium acetate in 90% DMF. 50 mg ofDCTA-SH (Example 8) in the triethylammonium form were added. The mixturewas stirred in the dark for 4 hours at 50° C. The DMF was removed underreduced pressure at 45° C. and the DNA was desalted by G-50 filtration.The degree of labelling was then determined by the use of radioactiveNi-63. On the average every 5.3 bases were labelled, by calculation.

EXAMPLE 13 4-Aminothiophenyl DCTA and 3-Aminothiophenyl DCTA ##STR46##

882 mg of 4-bromo-5-hydroxy-DCTA (2.0 μmole) and 376 mg of4-aminothiophenol, or 320 μl of 3-aminothiophenol, were dissolved in 2ml potassium carbonate (1M), and the mixture was stirred for 2 hours at90° C. under argon. The mixture was diluted with 50 μl oxygen-free H₂ Oand loaded to a 10 ml Dowex column. The column was washed with 0.1Macetic acid until the flow through was thiol free, and the product waseluted with 0.2 HCl. All thiol containing fractions were combined, andthe HCl was neutralized with KOH. The solution was stored at -40° C. Theyield determined by the thiol content was 92.7% (13-1) and 86.3% (13-2)respectively.

EXAMPLE 14 Labelling of BSA with 4-aminothiophenyl DCTA

When labelling with 4-aminothiophenyl DCTA (Example 13-1), the stock4-amino solution was 0.1M. 100 μl of the 4-aminothiophenyl DCTA solutionplus 25 μl 1M HCl plus 100 μl of 0.1M NaNO₂ were mixed and incubated for30 min. at 0° C. 50 μl of 1M K₂ HPO₄ were added to bring the pH to 6.7and a 50 μl aliquot was mixed with 1 ml of BSA (7.0 mg/ml) in 0.1MNABO₃. The mixture was incubated for one hour at 4° C. The diazoniumsalt was very labile. After 1 hour at 4° C., no coupling with β-naphtholwas observed; the ratio of labelling was 1.7.

The 3-aminothiophenyl DCTA was not used, but due to its high stabilityit will be a better reagent.

The BSA used here was previously heated for 15 minutes at 95° C. in pH3.5 to inactivate nuclease.

EXAMPLE 15 Labelling of BSA with DCTA Hydrazide

200 μl of hydrazide solution (6.7 umoles) plus 50 μl 1M HCl were cooledat 0° C. 7 μl of 1M NaNO₂ were added and incubated for 15 minutes at 0°C. 30 μl of ice-cold 2M Na₃ CO₃ were added to the solution to bring thepH to ca. 8.5-9.0. To 1 ml solution of BSA in 0.1M NABO₃ (7.0 mg/ml)were added 125 μl of the azide solution and the mixture was incubatedovernight at 4° C. The excess of azide was removed by G50 filtration.The BSA filtrate was diluted to 0.5 mg/ml and a 100 μl aliquot waslabelled with Ni. Total counts were 86,728 (6.9 1 moles). Assuming a MWof 68000 for BSA, the 0.5 mg are 7.3 μmoles 0.72 μmoles in the 100 mlaliquot corresponds to 9.4 DCTA molecules/molecule of BSA).

EXAMPLE 16 λ DNA Hybridization Using a Radio Cobalt-Containing DNA ProbeA. Preparation of DNA Carrying Terminal Poly(allylamine) dUTP

3.2 OD/260, 200 μg λDNA in 200 μl (0.01 buffer Tris-HCl pH 7.4, 0.001MEDTA) were mixed with 80 μl DNAse (diluted 1/5000 with 1 μg/μl BSA;0.005M MgCl₂). The mixture was incubated for 5 min. at 37° C., mixedwith 50 μl of 0.1M EDTA and heated for 15 min. at 65° C. to inactivatethe enzyme. The incubation mixture was loaded onto a 0.5 ml DEAE columnequilibrated in 0.2M KCl, and the column was washed with 5 ml 0.2M KCl.The DNA was eluted with 0.5M KOH. The 260 nm absorbing fractions werecombined and neutralized with acetic acid.

The incubation mixture contained in 3.0 ml: 0.2M cacodylate buffer pH7.3; 0.1M potassium acetate 1 mM dUTP-0.3 mM allyl-amino dUTP; 1 mMCaCl₂ ; 1 mM β-mercaptoethanol; digested DNA and 400 units of terminaltranferase. Incubation proceeded overnight at 37° C. 51.8% of thenucleotide triphosphates were terminally incorporated to the DNAfragments. The mixture was loaded onto a 0.5 ml DEAE column, washed with10 ml 0.2M KCl and the product was eluted with 1.5M LiCl 9.3 OD/260; 3.2OD of input=6.1 OD/260=244μg polymer.

B. Radio Labelling of the Allylamino groups

1 ml hydrazide-DCTA (Example 3) (16.4 mmoles) plus 200 μl 1M HCl werecooled at 0° C. 50 μl of cold 0.35M NaNO₂ were added under stirring andthe mixture was incubated for 15 minutes at 0° C. 100 μl of cold 2M K₂CO₃ were added to neutralize the acid, followed by the addition of 200μl 0.5M borate buffer pH 9.2. To this mixture, the "cold" DNA solution(1 ml) was added and incubated overnight at 0° C. The mixture was thenloaded on a 0.5 ml DEAE column (equilibrated in 0.2M KCl). The columnwas washed with 10 ml 0.2M KCl, and the product was eluted with 1.5MLiCl. Half of the OD/260 was eluted with this salt. The rest was elutedwith 1.5M LiCl in 0.2M acetic acid. Radioactive CoCl₂ was then added inaliquots.

Fr I: 8,800,000 cpm/μg.

Fr II: 92,600,000 cpm/μg

Fr I was then hybridized with DNA, as follows.

C. Hybridization C.1 Southern gels

DNA was restricted with Hind III, chromatographed and transferred by theSouthern blot technique to appropriate filters. As control were usedrestricted M13 phage. Cobalt-labelled DNA (Experiment 16.B, 2 μg), andcarrier DNA (1 mg) were dissolved in 5 ml of hybridization solution. Thecontrol and test samples were incubated with this solution at 42° C.overnight, then washed 3 times in buffer with 0.1% SDS at 55° C., airdried, and counted in standard toluene cocktail.

Results:

(1) M13 control: 7475 cpm.

(2a) λ test (a): 25,724 cpm.

(2b) λ test (b): 29,412 cpm.

The results indicate that, although background counts are high,hybridization using the cobalt labelled probe yielded about 3.5-4 timehigher counts.

C.2 Spot Hybridization

Spot hybridizations were carried out under overnight conditions asdescribed for C.1 above. Using the Co-labelled DNA probe it was possibleto detect 125 pg of λ DNA.

EXAMPLE 17 Preparation of Radio-Nickel Labelled Anti HCG Antibody

0.4 OD/₂₈₀ of anti human chorionic gonadotropin antibody+0.22 μmols ofhydrazide DCTA (Example 3) in 250 μl volume of 0.1M borate buffer wereincubated overnight at 0° C. The excess of DCTA hydrazide was removed byG50 filtration.

Filtrate

OD/₂₈₀ =0.376

OD/₂₈₀ =0.216

An aliquot (100 μl) containing the derivatized antibody was mixed with10 μmols Ni of specific activity 629,262 cpm/μmol. Counts bound to theprotein: 297,350 cpm=0.472 μmoles. (Assuming 1.4 OD/₂₈₀ for 1 mgantibody and a molecular weight of 150,000.). The 100 l aliquotcontained 0.0376 OD/₂₈₀ which is equal to 26.4 μg=0.175 μmoles ofantibody. 0.175 μmoles of antibody contained 0.472 μmoles Ni. Thus, 1mole antibody contained 0.472/0.176=2.60 moles Ni.

EXAMPLE 18 Recommended Procedure for DCTA-Labelling of Substances with aFree Alcohol Group, which are Soluble in Inert Solvents

Dowex 1-X2^(R) contains quaternary amino groups. These groups bind thecarboxy groups of DCTA-hydrazide very strongly to form the followingsalt (I): ##STR47##

Salt (I) is stable in 0.03M HCl and is displaced at higher (0.1M) HClconcentrations. It is reacted with NaNO₂ in 0.03M HCl to form azide(II): ##STR48##

By heating salt (II) in benzene the azide rearranges to form isocyanate(III): ##STR49##

The isocyanate group of (III) then reacts with alcohols at 80°-130° C.to form urethane (IV): ##STR50##

These urethanes are stable substances at physiological pH, and hydrolizeto form amines only with conc. acids or conc. alkali. The fixing of theDCTA to Dowex prevents, to a large extent, its --OH group reacting theisocyanate group of another molecule, and makes it possible to work inbenzene or toluene or other inert solvents where the DCTA azide isinsoluble.

After formation of the urethane (IV), the same is eluted from the Dowexwith cold LiCl in 50% ethanol. In the cold and at neutral pH notransesterification occurs.

EXAMPLE 19 Labelling of Ceramide

Ceramide is a diglyceride with two free alcohol groups.

10 mmoles of DCTA-hydrazide ((I) from Example 18) were added slowly to asuspension of Dowex AG-1-X2 in 50 ml H₂ O under stirring. The stirringwas continued for 15 minutes and the resin was washed on a Buchnerfunnel with 500 ml cold water. The resin was then suspended in 100 ml ofcold water. The resin was then suspended in 100 ml of cold 0.03M HCl,and the suspension was placed in an ice bath. 10 mmoles of solid NaNO₂were added under stirring in a period of 30 minutes, and the temperaturewas kept below 5° C. After the last NaNO₂ addition the suspension wasstirred for 15 minutes at the same termperature. The resin wassubsequently filtered through a cold Buchner funnel and washed with 100ml cold (-20° C.) ethanol. It was finally suspended in 100 ml of cold(-20° C.) ethanol and stirred for 30 minutes in an ice-salt bath at -20°C. The ethanol was removed by suction and the remaining traces ofethanol were removed at high vacuum. The dry resin was aliquoted underanhydrous conditions and stored in sealed ampoules at -70° C. withoutany loss of activity for at least 8 months.

To label the ceramide, 0.5 g of the azide resin were suspended in 15 mlabsolute benzene, 600 μmoles of ceramide were added and the suspensionwas heated for 30 minutes at 75° C. Subsequently, the benzene wasremoved in a vacuum, 15 ml absolute toluene were added, and thesuspension was stirred at 120° C. overnight under anhydrous conditions.

The resin was filtered, washed with ethanol to remove the traces oftoluene, and unreacted ceramide was then removed with 3 bed volumes of50% ethanol in H₂ O. The product was eluted with 0.6M LiCl in 50%ethanol/H₂ O. The fractions with DCTA activity were combined, and theceramide content was determined iodometrically. It was found to be 63.8%of the input.

EXAMPLE 20 A. General DCTA Labelling of Substances with a Cis-Diol Group

The diol is activated with cyanogen bromide in K₂ CO₃ solution, theexcess of cyanogen bromide is inactivated with triethanolamine, and theactivated diol is reacted with DCTA amine. If the substance is notsoluble in water, as is the case of digitoxin, the activation isperformed in diglyme/water.

B. Labelling of Digitoxin

65.4 μmoles of ³ H-digitoxin (1000 cpm/mg) were dissolved in 40 ml ofdiglyme and cooled to 4° C. An equal volume of 0.1M cold K₂ CO₃ wasadded and 300 mg of cyanogen bromide, in 2 ml of acetonitrile, wereadded at once, under stirring and cooling. The mixture was kept for 15minutes at 4° C. and the excess of cyanogen bromide was inactivated with5 ml of 2M triethanol amine hydrochloride (pH 1.2), resulting in a pH of8.5-9.0. 200 μmoles of DCTA-amine, dissolved in 2 ml H₂ O were added,and the mixture was incubated overnight at 4° C. and subsequently loadedonto a Dowex AG-1-X2 column. The column was washed with 50% ethanoluntil no counts were contained in the flow through, and the conjugatewas eluted with 0.6M LiCl by 50% ethanol. All radioactive fractions werecombined. Recovery of the radioactivity, which implies conjugateddigitoxin, was 57.6%.

EXAMPLE 21 Labelling of 4-Amino-1-Naphthol Phosphate with DCTA

The procedure developed to label 4-amino-1naphthol phosphate with DCTAapplies to all substances having a primary amino group.

A solution of 500 μmoles of 5-hydroxy-DCTA-4-B-thiopropionic acidhydrazide dissolved in 20 ml of 0.2M HCl was cooled to 0° C., and 500μmoles of solid NaNO₂ were added in a period of 10 minutes so that thetemperature remained below 5° C. 15 minutes after the last NaNO₂addition, the pH was brought to 8.5 with cold Na₂ CO₃ and 450 μmoles of4-amino-1-naphtol phosphate dissolved in 5 ml 1M NaHCO₃ were added. Themixture was incubated overnight at 4° C., diluted with 100 ml of H₂ Oand loaded onto a Dowex 1-X2 column in the acetate form.

The column was washed with 3 bed volumes 0.1M acetic acid, and theproduct was eluted with 0.6M LiCl in H₂ O. The DCTA-containing fractionswere combined, and the product was precipated with 3 bed volumes ofethanol at -20° C. overnight.

Yield: 86.4% of product: ##STR51##

The product is a substrate for alkaline phosphatase. By coupling theformed phenol with a diazonium salt a precipitate appears wich can belabelled with a radioactive metal. This constitutes a very sensitiveassay for alkaline phosphatase.

Having now full described this invention, it will be appreciated bythose of ordinary skill in the art that the same can be performed withina wide and equivalent range of structures, procedures and uses withoutaffecting the spirit or scope or any aspect of the invention or anyembodiment thereof.

What is claimed as new and desired to be secured by Letters Patent ofthe United States:
 1. A compound of the formula: ##STR52## where A³ isA² or a biopolymer selected from the group consisting of an oligo- orpolynucleotide, oligo- or polysaccharide, oligo- or polypeptide or asynthetic polymer, each of A² and said oligo-or polynucleotide, oligo-or polysaccharide, oligo- or polypeptide or synthetic polymer having mmodifiable reactive groups consisting of amino, hydroxy, 1,2-cis diOH,halide, aryl, imidazoyl, carbonyl, carboxyl, thiol or a residuecomprising an activated carbon; Z_(b) is fluorine, chlorine or bromine;and m is an integer from 1 to the total number of modifiable reactivegroups on A³.
 2. The compound of claim 1 wherein the biopolymer is apolypeptide which contains tyrosine, histidine or amino residues.
 3. Thecompound of claim 1 wherein the biopolymer is a polynucleotide whichcontains guanine bases.
 4. The compound of claim 3 wherein thepolynucleotide is DNA.
 5. The compound of claim 1 wherein A³ is apolypeptide or a polysaccharide.
 6. The compound of claim 1 wherein saidpolypeptide is a structural protein, an immunoglobulin, or an enzyme. 7.The compound of claim 6 wherein said immunoglobulin is a monoclonalantibody.
 8. The compound of claim 1 wherein said activated carbon insaid modifiable group is present in a tyrosine or histidine residue of apolypeptide, or on a guanine, cytidine or inosine base of apolynucleotide, and is capable of covalent coupling to a diazoniumcompound.
 9. The compound of claim 8 wherein said base is guanine. 10.The compound of claim 1 wherein A³ is a polynucleotide.
 11. The compoundof claim 10 wherein said polynucleotide is RNA or DNA.
 12. The compoundof claim 1 wherein Z_(b) is chlorine.
 13. The compound of claim 5wherein said reactive group is a tyrosine residue.